Using In vitro Translation

1
Using In vitro Translation

• Lisan Parker
• Dept of Pharmacology, Shieh Lab
• Methodology
• November 21, 2002

2
Making protein in a tube!

• Promega TNT T7-coupled transcription/translation
  system
• What is this??? Make a protein probe?
• Protocol
• Uses

3
Protein probe? (huh?)
Rabbit Reticulocyte lysate
Plasmid DNA
Amino acid mix -Methionine
RNasin
T7-RNA Polymerase
35S-Methionine
4
3. Run new protein probes on SDS-PAGE and
transfer to nitrocellulose
5
Key Points on TNT in vitro transcription/translat
ion

• Typically make 50-300ng protein (control)
• Can use 0.2-2 µg plasmid DNA
• Use canine pancreatic microsomal membranes for
  post-translational modifications (lower protein
  yield)
• Largest protein generated 176 kDa 1455 aa

6
Research Fields using TNT

• Cancer
• Virology
• Apoptosis
• Alzheimers
• Cardiology
• Transcription Activation

7
Why use this technique?

• What is it?
• What can it help you examine?
• What are the methods to investigate it?

8
Show me how to examine protein-protein
interaction

• Yeast-2 hybrid
• Immunoprecipitation
• Overlay Assay (Far Western)
• Interaction occurs on a membrane
• Pull-down Assay (Affinity Chromatography)
• Interaction occurs in solution

9
Making GST-fusion proteins
OR
Harvest Bacteria
Separate on SDS-PAGE
_at_ OD 0.6-0.8 add 0.1mM IPTG
Transfer to Nitrocellulose
10
Protein overlay assays
35S-protein X
Autoradiography or PhosphorImage
Western ?-GST
How do you identify the interacting proteins?
11
Example MUPP1 interaction with the Serotonin2C
receptor

• Where does the receptor touch or what region on
  MUPP1 does the receptor interact with?
• How much of the receptor carboxyl tail is
  necessary to interact with MUPP1?
• But what do we need to do first???
• hint if we make the receptor into a
  radiolabeled hot protein probe, what do we need
  to do to MUPP1?

12
(No Transcript)
13
WT 5-HT2C R tail selectively interacts with MUPP1
PDZ 9-11
Overlay
Western ?GST
Probe S35-WT 5-HT2C tail
14
SSV of 5-HT2C R necessary for MUPP1 interaction
Overlay
Western ?GST
Probe S35-GST PDZ 9-11
15
Pull-down Assay or Affinity Chromatography
Wash Elute Resolve on SDS-PAGE Transfer to
Nitrocellulose Develop
16
then
PhosphorImage or Autoradiograph
17
Example MUPP1 interaction with 5-HT2C R

• Does the 5-HT2C R interact with the same region
  of MUPP1 (PDZ 9-11) in an independent approach?
• What residues at the carboxyl tail of the 5-HT2C
  R are important for binding to PDZ 10 of MUPP1?

18
MUPP1 PDZ 9-11 exhibits enhanced binding for the
5-HT2C R tail
19
PDZ motif critical residues
Figure adapted from Becamel C et al. JBC (2001)
20
http//www.promega.com/cellfree/
http//www.promega.com/webclassroom/tntsem/tnttlk4
notes.pdf
21
References

• Promega (website) http//www.promega.com/webclassr
  oom/default.htm
• Promega (Life Science Catalog)
• Promega (Technical notes and bulletin)
• Adamski FR, Zhu M-Y, Bahiraei F, and B-H Shieh.
  (1998) Interaction of Eye Protein Kinase C and
  INAD in Drosophila. PNAS 273 17713-17719.
• Becamel C, Figge A, Poliak S, Dumuis A, Peles E,
  Bockaert J, Lubbert H, and C Ullmer. (2001)
  Interaction of Serotonin 5-Hydroxytryptamine Type
  2C Receptors with PDZ10 of the Multi-PDZ Domain
  Protein MUPP1. JBC 276 c12974-12982.
• Work in the Shieh laboratory